PROJECT SUMMARY Superbugs such as Methicillin-resistant Staphylococcus aureus (MRSA) is a global healthcare problem which exhibits high rates of morbidity and mortality amongst patients. While variant strains of bacterial resistance continue to emerge, the development of newer and more potent antibiotics has not kept pace, with urgent and burgeoning unmet need for several resistant infections including MRSA. It is thus imperative to find alternative solutions, such as to package current antibiotics into novel targeted delivery systems that can selectively deliver antibiotics at the site of the infection and smartly evade the bacterial barriers and for better therapeutic response with high safety. We for the first time, developed a folate decorated VAN nano-antibiotic (LVAN) that showed pronounced accumulation at the site of infection, leading to enhanced killing of MRSA strains in vitro and in vivo as compared to the commercially available VAN that forms the basis for further exploration. The goal of this project is to further develop and optimize a library of folate containing stimuli responsive nano-antibiotics with very high vancomycin (VAN) loading (~50% wt/wt) that will yield significant bactericidal activity against strains of MRSA while demonstrating high safety. Specifically, we will develop bacterial colony-responsive nano-antibiotic library synthesis for optimized killing of MRSA and selective delivery/release to improve infection treatment followed by determining the most optimal nano-antibiotic formulation by susceptibility testing and use of a validated PK/PD model against MRSA strains. These select few (1-2) nano-antibiotic formulation containing VAN will then be tested in vivo using a mouse thigh infection model against validated strains of MRSA followed by evaluation of biodistribution and kidney toxicity using a well characterized rat model. It is expected that the targeted nano-antibiotic formulation of VAN developed herein on systemic administration will show selective accumulation at the site of MRSA infection and exhibit controlled antibiotic release over an extended period of at least 72 hours improving efficacy. In addition, the liposomal formulation will be safer than commercially available VAN because of the expected lower dose exposure and the alternative route of elimination by the liver. With respect to positive outcomes, the work proposed in this application is expected to yield a safe and robust stimuli-responsive targeted nano-antibiotic formulation with high VAN loading that will have a major impact on improving the lives of MRSA infected patients. Since the formulation has a high drug loading with minimum side effects, the patient can be administered with therapeutic doses of VAN without any major adverse reactions or events. Furthermore, since the formulation utilizes FDA-approved excipients we do not see any outward difficulty in scale up and translation of the technology from the bench to the bedside.